SUMMARY Our understanding of bacterial physiology is limited by the fact that the majority of bacterial genes are uncharacterized. Even in the best studied model bacterium, Escherichia coli K-12, >1,200 genes are completely uncharacterized, and for many of the ?characterized? genes, very little functional information is known. Our ability to characterize gene functions is now outpaced by identification of new genes with unknown function. Hence, it is critical that we develop high-throughput methodologies to reliably assign gene function at a more rapid pace. Two such high-throughput methods, Chemical Genomics and SGA, have been developed for use in yeast, and subsequently applied in E. coli. However, these methods are labor-intensive, provide little information about essential genes, and are only readily applicable in species with one (for chemical genomics) or two (for SGA) deletion collections (deletion collections are available for only a few bacterial species). Furthermore, SGA is only applicable to species in which pairs of gene deletions can be easily combined. We will harness the combined power of CRISPR interference (CRISPRi) and next-generation sequencing technologies to redesign the Chemical Genomics and SGA approaches. Our preliminary data demonstrate the effectiveness of this approach in E. coli and establish CRISPRi in Salmonella. CRISPRi-based methods for Chemical Genomics and SGA have three major advantages over the established approaches. First, CRISPRi- based methods are far less labor intensive and can be applied more rapidly and more cheaply than the existing approaches. Second, CRISPRi-based methods are more effective for studying essential genes. Third, CRISPRi- based methods are readily applicable to a wide range of bacterial species, including species that lack deletion collections. We expect to identify groups of functionally related genes using each approach individually, and by combining data from both approaches. We expect that the relationships we identify between genes will be the basis of many future studies, as has been the case for Chemical Genomics and SGA data in yeast and E. coli. Moreover, our work will have a long-term impact by establishing these methods as facile, high-throughput tools for investigating gene function in a wide variety of bacterial species. The proposed work is highly significant because it provides a powerful solution to the major problem of identifying gene function. The proposed work is innovative because no prior studies have applied CRISPRi to Chemical Genomics or SGA. Moreover, there have been no previous high-throughput studies of gene function in Salmonella.